The τ-subunit of the clamp loader complex physically interacts with both the DnaB helicase and the polymerase III (Pol III) core α-subunit through domains IV and V, respectively. This interaction is proposed to help maintain rapid and efficient DNA synthesis rates with high genomic fidelity and plasticity, facilitating enzymatic coupling within the replisome. To test this hypothesis, CRISPR-Cas9 editing was used to create site-directed genomic mutations within the gene at the C terminus of τ predicted to interact with the α-subunit of Pol III. Perturbation of the α-τ binding interaction resulted in cellular and genomic stress markers that included reduced growth rates, fitness, and viabilities. Specifically, strains showed increased cell filamentation, mutagenesis frequencies, and activated SOS. fluorescence flow cytometry and microscopy quantified large increases in the amount of ssDNA gaps present. Removal of the C terminus of τ (I618X) still maintained its interactions with DnaB and stimulated unwinding but lost its interaction with Pol III, resulting in significantly reduced rolling circle DNA synthesis. Intriguingly, had the largest induction of SOS, high mutagenesis, and the most prominent ssDNA gaps, which can be explained by an impaired ability to regulate the unwinding speed of DnaB resulting in a faster rate of rolling circle DNA replication, inducing replisome decoupling. Therefore, τ-stimulated DnaB unwinding and physical coupling with Pol III acts to enforce replisome plasticity to maintain an efficient rate of synthesis and prevent genomic instability.

中文翻译：

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Tau 介导的 Pol III 合成和 DnaB 解旋酶解旋之间的偶联有助于维持基因组稳定性


夹钳装载复合物的 τ 亚基分别通过结构域 IV 和 V 与 DnaB 解旋酶和聚合酶 III (Pol III) 核心 α 亚基发生物理相互作用。这种相互作用被认为有助于维持快速有效的 DNA 合成速率以及高基因组保真度和可塑性，促进复制体内的酶促偶联。为了检验这一假设，使用 CRISPR-Cas9 编辑在 τ C 末端的基因内创建定点基因组突变，预计与 Pol III 的 α 亚基相互作用。 α-τ 结合相互作用的扰动导致细胞和基因组应激标记，包括生长速率、适应性和活力的降低。具体来说，菌株表现出细胞丝状化、诱变频率和激活的 SOS 增加。荧光流式细胞术和显微镜量化了单链 DNA 缺口数量的大幅增加。去除 τ (I618X) 的 C 末端仍然保持其与 DnaB 的相互作用并刺激解旋，但失去了与 Pol III 的相互作用，导致滚环 DNA 合成显着减少。有趣的是，具有最大的 SOS 诱导、高诱变和最显着的 ssDNA 缺口，这可以通过调节 DnaB 解旋速度的能力受损来解释，导致滚环 DNA 复制速度更快，从而诱导复制体解偶联。因此，τ 刺激的 DnaB 解旋以及与 Pol III 的物理耦合可增强复制体的可塑性，以维持有效的合成速率并防止基因组不稳定。